Using Engineered 2-O-Sulfotransferase to Determine the Activity of Heparan Sulfate C5-epimerase and Its Mutants

Abstract

Heparan sulfate (HS) is involved in essential physiological and pathophysiological functions. HS is a highly sulfated polysaccharide consisting of glucuronic acid (or iduronic acid) linked to glucosamine carrying various sulfo groups. Biosynthesis of HS involves sulfotransferases and an epimerase. The HS C5-epimerase converts glucuronic acid to iduronic acid. The method for determining the activity has been cumbersome due to the use of a site-specifically 3H-labeled polysaccharide substrate. Here, we report a two-enzyme coupling assay to determine the activity of C5-epimerase. HS 2-O-sulfotransferase (2OST) transfers the sulfo group to the 2-OH-position of glucuronic or iduronic acid. Unlike the wild type protein, 2-O-sulfotransferase mutant (2OST Y94I) transfers sulfate to the iduronic acid but not to the glucuronic acid. Thus, 2OST Y94I cannot sulfate N-sulfated heparosan, a polysaccharide containing glucuronic acid. Incubating N-sulfated heparosan with C5-epimerase converts some of the glucuronic acid to iduronic acid, thus becoming a substrate for 2OST Y94I. The susceptibility of the C5-epimerase-treated N-sulfated heparosan to 2OST Y94I modification directly correlates to the amount of the activity of C5-epimerase, proving that this two-enzyme coupling system can be used to assay for C5-epimerase. The method was further used to determine the activities of various C5-epimerase mutants. Our approach will significantly reduce the complexity for assaying the activity of C5-epimerase and facilitate the structural and functional analysis of C5-epimerase. Heparan sulfate (HS) is involved in essential physiological and pathophysiological functions. HS is a highly sulfated polysaccharide consisting of glucuronic acid (or iduronic acid) linked to glucosamine carrying various sulfo groups. Biosynthesis of HS involves sulfotransferases and an epimerase. The HS C5-epimerase converts glucuronic acid to iduronic acid. The method for determining the activity has been cumbersome due to the use of a site-specifically 3H-labeled polysaccharide substrate. Here, we report a two-enzyme coupling assay to determine the activity of C5-epimerase. HS 2-O-sulfotransferase (2OST) transfers the sulfo group to the 2-OH-position of glucuronic or iduronic acid. Unlike the wild type protein, 2-O-sulfotransferase mutant (2OST Y94I) transfers sulfate to the iduronic acid but not to the glucuronic acid. Thus, 2OST Y94I cannot sulfate N-sulfated heparosan, a polysaccharide containing glucuronic acid. Incubating N-sulfated heparosan with C5-epimerase converts some of the glucuronic acid to iduronic acid, thus becoming a substrate for 2OST Y94I. The susceptibility of the C5-epimerase-treated N-sulfated heparosan to 2OST Y94I modification directly correlates to the amount of the activity of C5-epimerase, proving that this two-enzyme coupling system can be used to assay for C5-epimerase. The method was further used to determine the activities of various C5-epimerase mutants. Our approach will significantly reduce the complexity for assaying the activity of C5-epimerase and facilitate the structural and functional analysis of C5-epimerase.